Vitamin B12 Nutriture of Chickens Fed Raw Soybean Meal1

Vitamin B12 Nutriture of Chickens Fed Raw Soybean Meal1 N. E. W A R D , 2 J. E. J O N E S , and D. V. M A U R I C E 3 Poultry

Science Department,

Clemson

University,

Clemson,

South

Carolina

29631

(Received for publication March 18, 1985)

1986 Poultry Science 6 5 : 1 0 6 - 1 1 3 INTRODUCTION

Raw soybean meal (RSM) requires processing or can be utilized only in limited amounts in poultry diets (Hill and Renner, 1963; Latshaw and Clayton, 1976) due to antinutritional factors (Liener and Kakade, 1980) and low digestibility of RSM protein (Nesheim and Garlich, 1966). Heat treatment is commonly used to deactivate protease inhibitors and denature the native soybean protein. The results of other processing techniques have been varied (De Valle, 1981). In search of an alternate method to enhance the nutritional value of RSM, addition of

1 Published with the approval of the Director of the South Carolina Agricultural E x p e r i m e n t Station as Technical C o n t r i b u t i o n No. 2 3 9 5 . 2 Taken in part from t h e dissertation s u b m i t t e d b y N. E. Ward t o the G r a d u a t e School, Clemson University, in partial fulfillment of the r e q u i r e m e n t s for the Ph.D. degree. 3 T o w h o m c o r r e s p o n d e n c e should be addressed.

106

vitamin B 12 was found to improve the biological value of RSM protein from 48.6 to 77.6% for rats, possibly through increased release of amino acids (Baliga et at, 1963). Supplementation of RSM diets with vitamin B n stimulated growth of rats, whereas other vitamins and minerals gave little or no response (Rackis, 1981). The addition of methionine to RSM diets fed to rats partially overcame the growth inhibition, particularly if Bn was included (Williams and Spray, 1973). Raw soy flour diets fortified with methionine, cysteine, or choline increased hepatic Bi 2 in rats, and the results suggested a depletion of sulfur amino acids (Edelstein and Guggenheim, 1971). Alterations in the microflora caused by RSM diets could be responsible for the deleterious effects of RSM, since bacterial synthesis, coupled with coprophagy, may represent a significant source of B 12 (Albert et al., 1980). In addition, RSM contains a goitrogenic factor (Liener, 1981), and low thyroid activity is known to impair the absorption of vitamin B 12 in rats (Okuda and Chow, 1967). In view of

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ABSTRACT E x p e r i m e n t s were c o n d u c t e d t o investigate a possible interference by raw soybean meal (RSM) with B 1 2 n u t r i t u r e of chickens. In E x p e r i m e n t 1, day-old chicks were fed B 1 2 -free isonitrogenous and equienergetic diets containing 0 or 40% RSM to d e t e r m i n e if RSM accelerated storage losses of B 1 2 . After 42 days, RSM decreased growth ( P < . 0 1 ) , decreased hepatic ( P < . 0 1 ) and blood ( P < . 0 5 ) c o n c e n t r a t i o n s of glutathione (GSH), and increased ( P < . 0 1 ) pancreas and liver weights. However, statistically significant differences d u e t o t r e a t m e n t were n o t d e t e c t e d in hepatic B 1 2 c o n c e n t r a t i o n , indicating t h a t RSM does n o t enhance B 1 2 turnover in chicks. T o ascertain the effect of RSM on B , 2 absorption, 9 Mg B 1 2 / k g diet was added t o diets containing 0 or 40% RSM and fed to chicks to 42 days of age. Raw soybean meal depressed growth ( P < . 0 0 1 ) and hepatic GSH (P-C01) and increased ( P < . 0 0 1 ) pancreas weights. T h e RSM had n o effect o n hepatic B 1 2 c o n c e n t r a t i o n , suggesting that RSM trypsin inhibition does n o t impair B I 2 absorption in chicks. A third e x p e r i m e n t was designed to test the hypothesis t h a t vitamin B l 2 stimulates egg production or feed intake of hens fed a diet with 27% RSM. Vitamin B I 2 - d e p l e t e d hens were fed RSM or heat-treated soybean meal (HSM) diets containing equivalent a m o u n t s of soybean protein, oil, and hulls. I lens received either 2 6 jug B 1 2 / k g BW per os or 13 Mg B 1 2 / k g BW intramuscularly twice within a 30-day period. Raw soybean meal reduced ( P < . 0 5 ) egg p r o d u c t i o n and feed intake, b u t B 1 2 failed t o affect either variable w h e n RSM or HSM constituted t h e major dietary protein source. B 1 2 - d e p l e t e d hens fed HSM gained weight u p o n B , 2 administration. These studes indicate t h a t vitamin B 1 2 nutriture of chicks is unaffected by dietary RSM, and t h a t B 1 2 does n o t stimulate egg p r o d u c t i o n or feed intake of h e n s fed RSM or HSM. T h e results of E x p e r i m e n t 2 s u p p o r t evidence for a single B 1 2 t r a n s p o r t protein in t h e intestinal tract of chickens. (Key words: vitamin B 1 2 , absorption and turnover, raw soybean meal, reduced glutathione, hepatic B 1 2 , chickens, feed intake, egg p r o d u c t i o n )

RAW SOYBEAN MEAL AND VITAMIN B 12

MATERIALS AND METHODS Animals and Husbandry. In Experiments 1 and 2, day-old New Hampshire x Single Comb White Leghorn (SCWL) chicks were wingbanded, weighed, and grown in thermostatically controlled brooders for 42 days under husbandry conditions previously described (Maurice et al., 1985). In Experiment 3, year-old SCWL commercial strain hens were individually maintained in 40 X 30 X 47 cm cages in an open-sided, naturally ventilated house. Vitamin Bi 2 -depleted hens were used to maximize sensitivity to small doses of the vitamin. The

hens were depleted of vitamin Bi 2 for 8 months and molted prior to the start of the experiment. Hens were inseminated with 50-jul pooled semen once a week, and hatchability of fertile eggs was measured. Water containers and cups were washed and disinfected at 7-day intervals to minimize microbial growth and vitamin Bi 2 synthesis. Diets. A 50% mixture of Govan and Braxton varieties of soybeans was ground twice through a 5-mm screen fitted to a Wiley mill. Processed soybean meal (HSM) was secured from a local feed mill. In Experiment 1, two equicaloric and isonitrogenous diets (Table 1) were formulated with HSM and RSM without the addition of vitamin Bi 2 . In Experiment 2, the same diets were fed with the addition of 9 jug vitamin B J 2 /kg diet. Diets were stored at 13 C. In Experiment 3, two vitamin B12-free diets were formulated to contain equivalent amounts of soybean protein, oil, and fiber (Table 1). Trypsin inhibitor content of the RSM was 33.5 mg/g compared to 4.4 mg/g for the HSM (Table 2). All diets contained a vitamin premix without added vitamin B 12 but otherwise met requirements (NRC, 1977). One-gram aliquots of the diets were periodically sampled for the determination of vitamin B i 2 concentration. Feed and water were supplied ad libitum in all experiments. Administration of Vitamin Bl2 to Hens. After a 3-week adjustment period to the diets, cyanocobalamin was administered either per os or intramuscularly at rates of 26 and 13 Mg/kg body weight, respectively. Approximately 50% of vitamin B 1 2 is absorbed from the intestinal tract (Edwards, 1969) hence, the difference in dosage. Treatments were spaced 24 days apart and the same route was assigned to hens for the second treatment. Measurements and Sample Collection. Individual body weights of chicks were recorded after 42 days. Blood was taken by frontal cardiac puncture into Li-heparin coated tubes and placed in an ice bath. Chicks were sacrificed by cervical dislocation, and livers were immediately excised, weighed, and immersed in liquid nitrogen. Livers were stored at —70 C and analyzed for reduced glutathione (GSH) within 72 hr. The pancreas was separated from the duodenum and weighed. Biochemical Analyses. Ground meal samples were delipidated with petroleum ether and a 1-g defatted sample was extracted with 10 m/W NaOH. Different amounts of the extract were

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the importance of intestinal trypsin activity for proper Bi 2 absorption (Allen et al., 1978) and the effects of fasting animals previously fed RSM (Ma'ayani and Kulka, 1968; Lyman et al., 1974; Hasdai and Liener, 1983), a large portion of the data of Edelstein and Guggenheim (1970) is consistent with impaired B 12 absorption or increased metabolic turnover of vitamin Bi 2 . Research on the effect of RSM on B 12 nutriture of chickens is limited. Underheated soybean meal was suggested to increase the requirement of vitamin B 12 for chicks (Frolich, 1954), although limited data were presented. Fisher et al. (1957) reported that the addition of 20 /Jg B I 2 /kg diet containing 60% solventextracted RSM produced the same effect as feeding processed soybean meal. These authors noted that feed intake was restored and satisfactory egg production was maintained in hens fed the RSM diets and postulated that vitamin B 12 enhanced the utilization of the amino acids or RSM protein. Collectively, these reports imply that vitamin B 12 might substitute, at least in part, for the use of heat treatment once the oil has been extracted, thus eliminating some expense in the manufacture of the meal. The present experiments were designed to determine if RSM increases the vitamin Bi 2 requirement of chickens, and if so, by what mechanism. The purpose of Experiment 1 was to determine whether RSM enhanced the loss of hepatic vitamin B 1 2 . Experiment 2 was designed to ascertain if malabsorption of vitamin B 12 (as measured by hepatic vitamin Bi 2 concentration) is induced by RSM diets. Experiment 3 tested the hypothesis that the adverse effects of RSM on egg production and feed intake are alleviated by vitamin B 1 2 .

107

108

WARD ET AL. TABLE 1. Composition and calculated analyses of diets1 (%) Experiment 1 and 2 HSM2

Calculated analyses Protein Metabolizable energy kcal/g Calcium Total p h o s p h o r u s Total sulfur a m i n o acids

57.84 29.66 5.00 4.50 2.30

RSM

HSM

58.63 20.30 40.00 4.52

2.18 .35

.40 .25 .05

.40 .25 .05

20.3 3.19 .88 .72 .66

20.3 3.19 .88 .72 .66

26.90 4.50

4.50 2.26 4.44 1.37 7.82 .13 .03 .40 .25 .05 .48

1.18 7.94 .11 .04 .40 .25 .05

16.0 2.95 3.47 .55 .64

16.1 2.95 3.55 .55 .64

1

Diets in Experiment 1 contained no vitamin B 1 2 ; the same diets were fed in Experiment 2 with the addition of 9 Mg vitamin B 12 /kg diet. Layer diets in Experiment 3 did not contain vitamin B 1 2 . 2

HSM = Heat-treated soybean meal, RSM = raw soybean meal.

3

Vitamin premix provided per kilogram diet: retinyl acetate, 8800 IU; cholecalciferol, 1115 IU; dl-ct-tocopheryl acetate, 11 IU; menadione sodium bisulfite complex, .55 mg; choline, 445 mg; nicotinic acid, 22 mg; riboflavin, 5.5 mg; calcium d-pantothenate, 5 mg; pyridoxine hydrochloride, 3 mg; thiamine mononitrate, 1.1 mg; folic acid, 550 Mg; d-biotin, 110 Mg"Mineral premix supplied per kilogram diet: Mn, 65 mg; Zn, 40 mg; Fe, 60 mg; Cu, 4 mg; I, 1 mg.

brought to 2 ml and trypsin inhibition measured (Hamerstrand et al, 1981). Urease activity was measured as the difference between the pH of a buffered urea solution and the pH of the blank (Smith and Circle, 1978). Whole blood GSH was determined by a colorimetric method (Beutler et al, 1963). Blood was hemolyzed in distilled water and hemolysate proteins separated by the addition of precipitating solution (1.67 g metaphosphoric acid, .2 g dipotassium EDTA, and 30 g sodium chloride in 100 ml distilled water). The filtrate was mixed with the color reagent and absorbance measured at 412 nm. Glutathione was used as the standard. For the analysis of hepatic GSH, 1 g liver was homogenized in 9.25 ml chilled 3% metaphosphoric acid solution, and the homogenate was treated as whole blood. Tissue and feed vitamin B 12 extraction was based on the protocol of Beck (1979). The

sample (1 g) was homogenized in 15 ml solution of sodium nitrite and sodium cyanide (5:2 g/liter, the pH was adjusted to 4.0 to 4.2 with HCl, and the mixture was boiled for 30 min in a

TABLE 2. Trypsin inhibitor activity and urease index of soybean meals RSM

Trypsin activity, mg/g 2 Urease index

HSM 1

Full fat

Defatted

4.4 .06

33.5 1.90

22.5 1.90

1 HSM = Heat-treated soybean meal, RSM = raw soybean meal. 2 Trypsin inhibitor was determined from the dilution that inhibited about 40% of the added tryp-

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Ingredients Yellow corn Soybean meal, 4 8 . 5 % Raw soybean meal Alfalfa meal S o y b e a n hulls Soybean oil Deflourinated p h o s p h a t e Limestone DL-Methionine Mono-lysine Salt Vitamin p r e m i x 3 Trace minerals" Filler, washed sand

Experiment 3

RSM

RAW SOYBEAN MEAL AND VITAMIN B 12

TABLE 3. Effect

of

feeding raw soybean

time as a split-plot. Variation among blocks within the treatment combination was used as the error term to test the main effects, and residual variation was used as the error term to test the response over time (Meade and Curnow, 1983). RESULTS

The purpose of Experiment 1 was to determine if RSM enhanced losses of stored vitamin B 12 in the liver. The RSM used in our experiments contained 33.5 mg/g trypsin inhibitor and urease index of 1.9 (Table 2). Cold delipidation reduced trypsin inhibitor activity by 33%. A summary of the effects of feeding a vitamin B12-free diet containing 40% RSM to chicks is presented in Table 3. After a 42-day feed period, growth and hepatic GSH were significantly depressed by dietary RSM. The RSM caused pancreatic hypertrophy, and this effect, as well as hepatic hypertrophy, was significant (P<.01). However, the source of soybean meal did not exert a significant effect on B 12 loss from the body as indicated by similar hepatic B 12 concentrations (P>.05). Experiment 2 was designed to determine if RSM caused malabsorption of Bi 2 as indexed by hepatic B 12 concentration. Summarized in Table 4 are the results of Experiment 2 in which vitamin Bj 2 was added to diets of identical composition as in Experiment 1. In agreement with the results of Experiment 1, RSM significantly depressed growth rate and hepatic GSH. An enlargement (P<.001) of the

m eal (RSM) in vitamin

B l : , -free diets to

chicks (Experiment

1)

RSM Response variaible

Body weight gain 0—6 wk, g2 Organ weight, % body weight2 Liver Pancreas Reduced glutathione, /^mol2 Blood/ml Liver/g Hepatic vitamin B 12> Mg/g3

0% 200

40%

114

SEM 1

P

± 11

<.01

2.71 .33

3.31 .62

.12 .04

<.01 <.01

3.34 3.76

2.28 2.45

.30 .28

<.05 <.01

.05

>.05

.45

1

SEM = Standard error of the mean.

2

Based on 8 chicks/pen and 3 blocks/treatment.

3

Based on 3 chicks/pen and 3 blocks/treatment.

.5 3

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hood under dim light. Sodium sulfate (200 mg/ml) was dissolved in the clear filtrate, then extracted thrice with 1/10 volume of benzyl alcohol per extraction. The combined extracts were mixed with 5/10 volume chloroform and extracted thrice with 1/10 volume water. Vitamin B 12 was determined by radioassay (RIA Products, Inc., Waltham, MA 02154) using purified hog intrinsic factor as binder to eliminate the measurement of vitamin B 12 analogues. The aqueous extract was mixed with labeled vitamin B 12 solution, incubated, then the binder solution was added and the mixture incubated in the dark at room temperature. The unbound fraction was adsorbed on charcoal, and the bound vitamin B 1 2 in the supernatant was counted in a gamma counter (Gamma4000, Beckman Instruments, Inc., Irvine, CA 92713). Quantitation was based on standards within the range of 50 to 2000 pg/ml. Experimental Design and Statistical Analyses. Treatments in Experiments 1 and 2 (0 vs. 40% RSM) were arranged in a randomized, complete block design with three blocks/ treatment. Each tier of the battery constituted a block, and within a block, each treatment was allocated at random to a pen of 8 chicks, which comprised the experimental unit. In Experiment 3, a randomized, complete block was used with a factorial arrangement of diets (RSM vs. HSM) and route of vitamin B 12 administration (per os vs. intramuscular). Data obtained at zero time and after administration of vitamin B 12 were analyzed by analysis of variance with

109

110

WARDETAL. TABLE 4. Effect of feeding raw soybean meal (RSM) in diets with vitamin B 1 2 (Experiment 2) RSM

Response variable

0%

Body weight gain 0 - 6 wks, g2 Pancreas weight, % body weight2 Hepatic reduced glutathione, jumol/g2 Hepatic vitamin B 12 ,/jg/g 3

40%

291

123

.38 3.27 .58

1

SEM = Standard error of the mean.

2

Based on 8 chicks/pen and 3 blocks/treatment.

3

Based on 3 chicks/pen and 3 blocks/treatment.

P

±13 ± .03 ± .24 ± .06

<.001 <.001 <.01 >.05

significantly depressed. A possible explanation could be that RSM places a strain on sulfur amino acid metabolism (Kwong and Barnes, 1963) by stimulating excessive secretion of pancreatic enzymes (Liener and Kakade, 1980) rich in sulfur amino acids. Limited methionine and cysteine diminishes hepatic GSH in rats (Glazenburg et al., 1983). This functional deficiency may have been further aggravated by the low digestibility of raw soybean protein (Nesheim and Garlich, 1966). Our results indicate that plasma GSH may be used as a response variable to evaluate RSM in the diet. Hens fed 27% RSM consumed less feed, were unable to maintain mature body weight, and produced fewer eggs. The latter responses are a sequel to a depression in appetite, and the mechanism by which RSM decreases feed consumption probably originates with the trypsin inhibitors. Trypsin inhibition stimulates secretion of cholecystokinin (Baile et al., 1983), and injected cholecystokinin reduces feed intake in hens (Savory and Gentle, 1980). Dietary propionic acid is known to speed B 12 depletion in hens (Ward et al., 1983) and chicks (Rys and Koreleski, 1974) by enchancing the need for the B 12 -dependent isomerase

DISCUSSION The present study was designed to test a postulated antagonism by RSM on vitamin B(2 nutriture of chickens, but all experiments consistently failed to detect such a relationship. Growth retardation and pancreatic enlargement were present in chicks fed 40% RSM, and these effects are consistent with previous observations (Saxena et al, 1963). In addition, GSH concentration in whole blood and liver was

TABLE 5. Hatchability of fertile eggs from hens with (+) and without (—) dietary vitamin B12 Month

B n (+)

B „ (-) (% hatchability) —

February June October

90.1 91.0 93.4

92.3 7.5 0

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pancreas was present in those chicks consuming the RSM diets. Hepatic Bi 2 concentration was not affected by RSM (P>.05), suggesting that B] 2 absorption was not hampered by trypsin inhibition. Experiment 3 was conducted to test the hypothesis that vitamin B 12 reduced or eliminated the adverse effects of RSM on egg production and feed intake of hens. The diets were periodically assayed for the presence of B 12 contamination, but none was detected in the B 12 deficient diet. The diet with supplemental vitamin Bi 2 assayed 14 ± 2.19 /ig/kg. After 8 months, fertile egg hatchability was 0%, indicating that a severe state of Bi 2 deficiency had been attained (Table 5). The RSM decreased (P<.05) egg production in the hens by about 40%, while feed intake was decreased (P<.05) by approximately 18%. No effect (P>.05) on egg production or feed intake was recorded for the 14-day period after B 12 administration (Table 6). Treatments were repeated 24 days later, and again B 12 failed (P>.05) to favorably affect either variable when RSM constituted the major dietary protein source. Hens consuming HSM diets gained weight after B 12 dose, but this effect was not apparent in those hens receiving the RSM diets.

.74 2.13 .63

SEM1

Ill

RAW SOYBEAN MEAL AND VITAMIN B.

TABLE 6. Effect of vitamin B12 administration to B ^-depleted hens fed heated (HSM) and raw soybean meal (RSM) > RSM

HSM i/m 2

Oral

Oral

i/m

SEM

5.4 4.5 4.8

± 1.1

Egg production, eggs/14 days 9.2 9.6 9.3

Pre B 1 2 dose Post 1st B, 2 Post 2nd B „ HSM vs. RSM

1719 1736 1743

Pre B n dose Post 1st B 12 Post 2nd B 12 HSM vs. RSM

57.7 57.3 56.3

a

9.3

8.3 10.9 8.6

a

5.3 4.5 4.9 4.9

1645 1666 1665

1567 1554 1525

1696 a

60.3 59.3 57.3 58.0

a

b

±

1431 1431 1407 1486 b

47.8

b

± 88 ± 36

50.2 49.7 47.4

47.1 46.8 45.8

.44

± 2.4 ± 1.0

' Means with different superscripts are significantly (P<.05) different.

1

Data presented by 14-day periods.

2

i/m = Intramuscular injection.

for methylmalonyl CoA metabolism. Increased requirement for endogenously derived methionine likewise might be expected to stress B12 stores. Edelstein and Guggenheim (1971) noted that methionine supplementation to raw soy diets increased hepatic B 12 of rats, although diets deficient in methionine did not affect tissue methylcobalamin (Linnell et al., 1983). The data of Experiment 1 are in agreement with the latter report (Linnell et al., 1983) in that B12 losses were not accelerated when metabolic methionine was presumably limited. A different approach was taken in Experiment 2, in which the diets fed in Experiment 1 were supplemented with 9 jUg B 12 /kg diet. Hepatic Bn was determined as an index of B 12 absorption. The RSM had no effect on hepatic B 1 2 , which contrasts with the speculation that trypsin inhibition might impair B [ 2 absorption in chickens. Earlier work (Allen et al., 1978; Marcoullis, et al., 1980) designated an essential role for intestinal trypsin for normal B 12 absorption in humans. Bj 2 preferentially binds to R proteins, and not intrinsic factor, in the acid milieu of the stomach (Allen et al., 1978), which transports the vitamin to ileal receptors specific only for intrinsic factor. The data in Experiment 2 are consistent with

the report that only one protein is involved in the intestinal transport of vitamin B 12 in chickens (Sonneborn and Hansen, 1970) and that transfer of the vitamin from one protein to another does not take place in the intestine. Indeed, the absence of a double protein B 1 2 transport system in chickens would nullify the importance of trypsin activity for proper Bj 2 absorption. Experiment 3 tested the hypothesis that B 1 2 ameliorates the adverse effects of RSM on egg production and feed intake by hens. Administration of vitamin B 1 2 , at a level of about 45-fold higher than NRC (1977) by injection or per os, did not elicit a favorable response in feed intake or egg production. These data contradict those of Fisher et al. (1957). Vitamin Bj 2 may exert an advantageous effect on soybean meal that has been partially processed, although a mechanism is not apparent. Both Fisher et al. (1957) and Frolich (1954) fed soybean meal that was defatted and heated to volatilize the solvent, whereas in our experiments, whole soybeans were ground and fed without additional processing. We did record an increase in body weights upon B [ 2 administration to hens fed the HSM diets. Vitamin B 12 deficiency in hens sig-

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Pre B 1 2 dose Post 1st B 12 Post 2nd B 12 HSM vs. RSM

112

WARD ET AL.

Frolich, A., 1954. Relation between the quality of soybean oil meal and the requirements of vitamin B 12 for chicks. Nature 173:132-133. Glazenburg, E. J., I.M.C. Jekel-Halsem, E. Schjoltens, A. J. Baars, and G. J. Mulder, 1983. Effects of variation in the dietary supply of cysteine and methionine on liver concentration of glutathione and "active sulfate" (PAPS) and serum levels of sulfate, cysteine, methionine and taurine: relation to the metabolism of acetaminophen. J. Nutr. 113:1363-1373. Hamerstrand, G. E., L. T. Black, and J. D. Glover, 1981. Trypsin inhibitor in soy products: modiThat the feeding of RSM to chicks would fication of the standard analytical procedure, enhance Bi 2 turnover or impair B 12 absorption Cereal Chem. 58:42-45. was the original supposition of this study. Yet, Hasdai, A., and I. E. Liener, 1983. Growth, digestibility and enzymatic activities in the pancreas neither effect was detected, and furthermore, and intestines of hamsters fed raw and heated soy vitamin B x 2 did not alleviate the aversion to flour. J. Nutr. 113:662-668. RSM or enhance egg production of hens. Our Hill, F. W., and R. Renner, 1963. Effects of heat data suggest that pancreatic trypsin does not treatment on the metabolizable energy value of play a role in the absorption of vitamin B J 2 in soybeans and extracted soybean flakes for the hen. J. Nutr. 80:375-380. the domestic chicken. Additional research is needed to define the exact mechanism for Hong, S. C , and D. L. Layman, 1984. Effects of leucine in in vitro protein synthesis and degradavitamin Bi 2 intestinal transport in aves. tion in rat skeletal muscles. J. Nutr. 114:1204— 1212. Kwong, E., and R. H. Barnes, 1963. Effect of soybean REFERENCES trypsin inhibitor on methionine and cysteine Albert, M. J., V. I. Mathan, and S. J. Baker, 1980. utilization. J. Nutr. 81:392-398. Vitamin B u synthesis by human small intestine Latshaw, J. D., and P. C. Clayton, 1976. Raw and bacteria. Nature 283:781-783. heated full fat soybeans in laying diets. Poultry Allen, R. H., B. Seetharam, E. Podell, and D. H. Sci. 55:1268-1272. Alpers, 1978. Effect of proteolytic enzymes on Liener, I. E., 1981. Factors affecting the nutritional the binding of cobalamin to R protein and quality of soya products. J. Am. Oil Chem. Soc. intrinsic factor. J. Clin. Invest. 61:47—54. 58:406-415. Baile, C. A., M. A. Della-Fera, and C. L. McLaughlin, Liener, I. E., and M. L. Kakade, 1980. Protease 1983. Hormones influencing feed intake. Georgia inhibitors. Pages 7—71 in Toxic Constituents of Nutr. Conf., Atlanta, GA. Plant Foodstuffs. I. E. Liener ed. Academic Press, New York, NY. Baliga, B. R., H. N. Bhagavan, and R. Rajagopalan, Linnell, J. C , M. J. Wilson, Y. B. Mikol, and L. A. 1963. Vitamin B 12 and the nutritive value of raw Poirier, 1983. Tissue distribution of methylsoybean. Indian J. Med. Res. 51:749—757. cobalamin in rats fed amino acid-defined, methBeck, R. A., 1979. Essential prerequisites for the yl deficient diets. J. Nutr. 113:124-130. analysis of cyanocobalamin in biochemically complex samples using radiometric competitive Lyman, R. L., B. A. Olds, and G. M. Green, 1974. binding assays. Pages 675—679 in Vitamin B u , Chymotrypsinogen in the intestine of rats fed B. Zagalak and W. Friedrich, ed. Walter de soybean trypsin inhibitor and its inability to Gruyter and Co,. suppress pancreatic enzyme secretions. J. Nutr. 104:105-110. Beutler, E., O. Duron, and B. M. Kelly, 1963. ImMa'ayani, S., and R. G. Kulka, 1968. Amylase, proproved method for the determination of blood carboxypetidase and chymotrypsinogen in panglutathione. J. Lab. Clin. Med. 61:882-888. creas of chicks fed raw or heated soybean diet. J. De Valle, F. R., 1981. Nutritional qualities of soya Nutr. 96:363-367. protein as affected by processing. J. Am. Oil Marcoullis, G., Y. Parmentier, J. Nicolas, M. Jimenez, Chem. Soc. 58:419-429. and P. Gerard, 1980. Cobalamin malabsorption Edelstein, S., and K. Guggenheim, 1970. Causes of the due to non-degradation of R proteins in the increased requirement for vitamin B 12 in rats human intestine. J. Clin. Invest. 66:430—440. subsisting on an unheated soybean flour diet. J. Nutr. 100:1377-1382. Maurice, D. V., J. E. Jones, M. A. Hall, D. J. Castaldo, J. E. Whisenhunt, and J. C. McConnell, 1985. Edelstein, S., and K. Guggenheim, 1971. Effect of Chemical composition and nutritive value of sulfur amino acids and choline on vitamin B 1 2 naked oats (Avena nuda L.) in broiler diets. deficient rats. Nutr. Metab. 13:339—343. Poultry Sci. 64:529-535. Edwards, H. M., Jr., 1969. The absorption of " C o or 60 Meade, R., and R. N. Curnow, 1983. Statistical Co-B ,j by the laying hen and mature rooster. Methods in Agriculture and Experimental Biology. Poultry Sci. 48:414-420. Chapman and Hall, New York, NY. Fisher, H., D. Johnson, Jr., and S. Ferdo, 1957. The National Research Council, 1977. Nutrient Requireutilization of raw soybean meal protein for egg ments of Domestic Animals. 1. Nutrient Reproduction in the chicken. J. Nutr. 61:611—621. nificantly reduces plasma leucine, and in vitro studies have confirmed a necessity of B 12 for leucine synthesis in the domestic fowl (Ward et al., 1985). Enzootic marasmus has been reported as a B 12 deficiency symptom in some species (Underwood, 1977), and the reported association between leucine and nitrogen losses (Sherwin, 1978) and protein synthesis in skeletal muscle (Hong and Layman, 1984) may contribute to an improvement in weight gain.

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RAW SOYBEAN MEAL AND VITAMIN B u

turnover and metabolic response to leucine. J. Clin. Invest. 61:1471-1481. Smith, A. K., and S. J. Circle, 1978. Pages 454-455 in Soybeans-Chemistry and Technology. The AVI Publishing Co., Inc., Westport, CT. Sonneborn, D. W., and H. J. Hansen, 1970. Vitamin B 12 binders of chicken serum and chicken proventriculus are immunologically similar. Science 168:591-592. Underwood, E. J., 1977. Cobalt. Trace Elements in Human and Animal Nutrition. Academic Press, New York, NY. Ward, N. E., J. E. Jones, and D. V. Maurice, 1985. Vitamin B u -dependent biosynthesis of leucine in chickens. Page 55 in Proc. South. Poultry Sci. Soc. Ward, N. E., D. V. Maurice, and J. E. Jones, 1983. Vitamin B 12 status of hens fed propionic acid. Poultry Sci. 62:1521. (Abstr.) Williams, D. L., and G. H. Spray, 1973. The effects of diets containing raw soya-bean flour on the vitamin B, 2 status of rats. Br. J. Nutr. 29:57—63.

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quirements for Poultry. Natl Acad. Sci., Washington, DC. Nesheim, M. C , and J. D. Garlich, 1966. Digestibility of unheated soybean meal for laying hens. J. Nutr. 88:187-192. Okuda, K., and B. F. Chow, 1967. The thyroid and absorption of vitamin B 12 in rats. Endocrinology 68:607-610. Rackis, J. J., 1981. Significance of soya trypsin inhibitors in nutrition. J. Am. Oil Chem. Soc. 58:495-501. Rys, R., and J. Koreleski, 1974. The effect of dietary propionic acid on the requirement of chicks for vitamin B 1 2 . Br. J. Nutr. 31:143-146. Savory, C. J., and M. J. Gentle, 1980. Intravenous injections of cholecystokinin and caerulein suppress food intake in domestic fowls. Experienda 36:1191-1192. Saxena, H. C , L. S. Jensen, and J. McGinnis, 1963. Influence of age on utilization of raw soybean meal by chickens. J. Nutr. 80:391-396. Sherwin, R. S., 1978. Effect of starvation on the

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